The primary objective of this project is to define molecular and biochemical mechanisms involved in eukaryotic DNA synthesis. To approach this problem, we are investigating adeno-associated virus (AAV) DNA replication in both in vivo and in vitro systems. We have shown that AAV DNA synthesis can be initiated in vitro in cell-free extracts and that replicating forms that correspond to those identified in vivo can be synthesized with either endogenous or exogenously added templates. Recently, we have identified two DNA-associated proteins that may play an important role in the required processing of self-primed unit length hairpin and concatemeric intermediates of DNA replication. The observed mode of AAV DNA replication may serve as a model for telomere replication in eukaryotes. Among methods used are affinity chromatography, gel-electrophoresis, DNA sequence analysis, and restriction cleavage of DNA molecules.